CN117777286A - Antibody A4D7 targeting procalcitonin and application thereof - Google Patents

Abstract

The invention provides a procalcitonin-targeted antibody A4D7 and application thereof. The amino acid sequences of the light chain and the heavy chain of the antibody A4D7 are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2. The antibody is used for developing a lateral flow immunochromatography detection reagent. The antibody has high sensitivity and good specificity, and has wide application prospect in the immunodiagnosis field.

Description

Antibody A4D7 targeting procalcitonin and its application

Technical Field

The present invention relates to the technical field of immune detection, and specifically to an antibody A4D7 targeting procalcitonin and its application.

Background technique

Procalcitonin (PCT) is a non-hormonally active glycoprotein composed of 116 amino acids and the precursor of calcitonin (CT). PCT was discovered in tumor marker research in the 1980s. In the 1990s, during studies on markers of acute lung injury, it was found that the serum PCT index increased sharply in patients with sepsis and septic shock. This topic was introduced to discuss whether there is a relationship between PCT and infection. Follow-up studies have shown that PCT can be used as a serum marker for severe bacterial infections, especially sepsis. It is an important indicator for early differential diagnosis of systemic inflammatory response and can be used to judge Important indicators of disease severity, treatment efficacy and prognosis.

The clinical application of PCT in immune regulation as an inflammation detection indicator:

As an immunomodulatory protein, PCT is one of the most commonly used markers for detecting inflammation. PCT induction is strictly regulated by the body, and its activation steps are highly correlated in time and function. This correlation may be used to reflect the severity of the disease. Under physiological conditions, PCT is synthesized by thyroid parafollicular cells. In the presence of bacterial infection, cells in other parts of the body (such as monocytes and neuroendocrine cells) activate the induced expression of the CALC-I gene corresponding to procalcitonin under stimulation of cytokines, resulting in the continued expression of PCT and freed. In addition, PCT is very stable in vivo and in vitro, easy to detect, and its concentration is not easily affected by the body's immunosuppressive state. For this reason, PCT is considered a biomarker that plays an important role in the early detection of systemic inflammatory response, and is widely used in the differential diagnosis, disease judgment and prognosis of a variety of clinical symptoms including bacterial infectious diseases. Assessment, including but not limited to major trauma (accidental trauma, surgery, burns), multiple organ dysfunction, severe pancreatitis or severe liver or kidney disease, and early neonatal period. In addition, non-infectious elevated PCT may be related to factors such as severe tissue damage, elevated plasma pro-inflammatory cytokine levels, severe organ environment disorders, or paraneoplastic syndromes. PCT has high sensitivity and specificity for distinguishing infectious and non-infectious fevers (such as tumor fever, drug fever). Lymphoma patients are prone to serious infections due to immune dysfunction and agranulocytosis after chemotherapy. Studies have shown that PCT levels in neutropenic patients with obvious infection and fever are significantly increased, while PCT levels in patients with focal infection or unexplained fever do not change significantly, which provides early diagnosis of bacterial bloodstream infections and avoids overuse of antibiotics. The application provides a reference.

Standardized testing methods for PCT:

The half-life of PCT is 25-30 hours. It has good in vitro stability and is easy to detect. The PCT concentration decreases by about 12% after 24 hours of blood collection at room temperature. Only a 6% reduction when stored at 4°C. Therefore, if blood cannot be tested immediately after collection, the sample must be cryopreserved. At present, PCT clinical detection methods mainly include chemiluminescence immunoassay, radioimmunoassay, colloidal gold method, transmission immunoturbidimetry, time-resolved fluorescence immunoassay technology, etc. The basic principle is the immune binding reaction of antigen and antibody.

The detection method of the lateral flow rapid diagnostic kit can usually only provide qualitative results and is not suitable for specific pathogens (such as certain bacteria, mycoplasmas, viruses, etc.) that require precise quantitative results to diagnose the infection. The lateral flow rapid diagnostic kit based on time-resolved fluorescence immunoassay technology, under the premise of using specific instruments and using the above-mentioned (such as new designs such as rare earth element chelates) specific antibody chemical labeling, has the characteristics of high sensitivity, convenient operation, high degree of automation, wide standard curve, no interference from natural sample fluorescence, simple and stable preparation of markers, and no radioactive pollution, and can be widely used for rapid detection or diagnosis. Through the immune response, it is possible to consider using quantitative or semi-quantitative methods to estimate the concentration of the labeled antibody and to deduce the concentration of the bound target antigen, so that theoretically rapid diagnosis and precise (semi-)quantification of specific pathogenic microorganisms can be achieved.

In any case, the lateral flow rapid diagnostic kit has reached the general standards of sensitivity, stability, economy and ease of use. Usually, it does not require the equipment required for rapid diagnosis, so it has important and extensive practical value. . These advantages of lateral flow immunochromatography rapid diagnostic kits are particularly important for units and regions that lack professional equipment and untrained technical personnel. For units and regions with professional equipment and trained technical personnel, the corresponding products The new design can still meet fine quantitative or semi-quantitative requirements.

Contents of the invention

The purpose of the present invention is to provide an antibody A4D7 targeting procalcitonin and an application thereof.

In order to achieve the purpose of the present invention, in the first aspect, the present invention provides an antibody A4D7 targeting procalcitonin. The amino acid sequences of the light chain hypervariable regions CDR-L1, CDR-L2 and CDR-L3 of the antibody are respectively RSSDSLLHSNGITYLF, QMSALAS and AQNLHLPWT. The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain hypervariable region of antibody M1301 are SDYSWE, YIDFRGSTKYNPSLRS and RGSFRY respectively.

The amino acid sequences of the light chain and heavy chain of the antibody are shown in SEQ ID NO: 1 and 2 respectively.

In a second aspect, the present invention provides the use of the antibody in preparing procalcitonin detection reagents or kits.

In a third aspect, the present invention provides a procalcitonin detection reagent or kit prepared from the antibody.

In a fourth aspect, the present invention provides the use of the antibody in preparing procalcitonin immunochromatographic detection test strips or kits.

In a fifth aspect, the present invention provides the use of the antibody in preparing a procalcitonin lateral flow immunochromatography diagnostic kit.

In a sixth aspect, the present invention provides a procalcitonin immunochromatographic detection test strip, which includes a sample pad, a binding pad, an NC membrane, an absorbent pad and a bottom plate. The reaction membrane is provided with a detection line and a quality control line. The sample pad, bonding pad, NC film and water-absorbent pad are adhered to the bottom plate in sequence.

Wherein, the binding pad is coated with immune (fluorescence) microsphere-coupled antibody F4B4 targeting procalcitonin; the detection line is coated with antibody A4D7, and the quality control line is coated with goat anti-mouse polypeptide anti.

The amino acid sequences of the light chain and heavy chain of antibody F4B4 are shown in SEQ ID NO: 3 and 4 respectively.

Preferably, the concentration of antibody F4B4 coated on the binding pad is 10-15 μg/mL.

Preferably, the concentration of antibody A4D7 coated on the detection line is 1.5-2 mg/mL.

Preferably, the material of the sample pad and the water-absorbent pad is cellulose, and the material of the bonding pad is glass fiber.

In the seventh aspect, the present invention provides a qualitative and (semi-)quantitative rapid detection method for procalcitonin (including non-diagnostic purposes). The detection method is a double-antibody sandwich method, and the first antibody used is a procalcitonin-targeting method. Antibody F4B4 (the amino acid sequences of its light chain and heavy chain are shown in SEQ ID NO: 3 and 4 respectively), and the second antibody used is antibody A4D7.

In the present invention, antibodies F4B4 and A4D7 respectively bind to different epitopes of procalcitonin.

In the present invention, the corresponding antigen amino acid sequence is an amino acid sequence that has been adjusted or modified accordingly, and the modified materials include but are not limited to one or more of nanomaterials, fluorescent materials, enzymes, biotin, and proteins. It is beneficial to apply the adjusted or modified amino acid sequence to the detection of procalcitonin, the design of immune antigen for procalcitonin, etc.

Through the above technical solutions, the present invention has at least the following advantages and beneficial effects:

(I) The present invention provides an antibody A4D7 that targets and recognizes procalcitonin, and uses it to construct a lateral flow immunochromatography rapid diagnostic kit. The antibody has high specificity and high affinity characteristics, and one or more of the amino acid sequences are used as the core. It is applied to the design of therapeutic targets for procalcitonin, and a therapeutic preparation for procalcitonin can be designed. The therapeutic target design includes but is not limited to the target design of therapeutic antibodies.

(2) The antibody can not only be effectively used for quantitative and/or qualitative detection of procalcitonin, but can also be used for various detection methods such as ELISA detection, immunochemiluminescence detection, and immunofluorescence detection, with a wide range of applications and applicability. powerful.

(3) The advantage of the present invention's rapid diagnostic kit based on lateral flow immunochromatography is that it has a lower detection limit and higher sensitivity; and the selected antibodies specifically target antigenic epitopes with highly conserved amino acid sequences, reducing detection costs. , shorten the detection time and improve the detection efficiency. Lateral flow immunochromatography rapid diagnostic kit is a detection technology with important application value, which can achieve the common standards required for rapid diagnosis such as sensitivity, stability, economy, user-friendliness and no equipment required. These advantages of the lateral flow immunochromatography rapid diagnostic kit are of great significance, especially for units or regions that lack professional equipment and well-trained technical personnel.

Description of the drawings

FIG. 1 is the result of protein electrophoresis using purified recombinant PCT protein in a preferred embodiment of the present invention.

Figure 2 is a graph showing the binding curves of antibody A4D7 with recombinant procalcitonin antigen at gradient concentrations in a preferred embodiment of the present invention, wherein 0.5 μg (/mL) is the binding curve of 50 ng of antigen per well (100 μL per well), and 1 μg (/mL) is the binding curve of 100 ng of antigen per well (100 μL per well).

Figure 3 shows the results of the paired screening experiment of monoclonal antibodies in the preferred embodiment of the present invention; where A is A4D7 paired with F4B4, B is A4D7 paired with A8E5, and C is A4D7 paired with C6H1.

Figure 4 shows the stability investigation results of antibody A4D7 in the preferred embodiment of the present invention.

Figures 5 and 6 are the results of testing (different dilutions) the lateral flow immunochromatography rapid test reagent developed based on the antibody of the present invention using the calcitonin pro-solution standard substance in a preferred embodiment of the present invention; wherein Figure 5 is the analysis result without batch division, and Figure 6 is the analysis result in batches.

Detailed ways

The following examples are used to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products. Example 1 Preparation of monoclonal antibody-producing hybridomas

1. Design, preparation and carrier coupling of procalcitonin protein antigen

According to the relevant information of the procalcitonin gene on GenBank (NM_001741.3, SEQ ID NO: 5), the amino acid sequence of procalcitonin (SEQ ID NO: 6) was obtained. After immunogenicity, hydrophilicity and surface accessibility analysis, the polypeptide sequence of 141 amino acids in the CDS region of procalcitonin was finally selected as the antigen (Figure 1). The antigen procalcitonin was synthesized by self-expression (the full-length gene was cloned into the expression vector pET20b).

2. Immunized mice

Mix the synthetic antigen procalcitonin and Freund's complete adjuvant at a ratio of 1:1 into 500 μL and emulsify it, then subcutaneously inject 3 female Balb/c mice aged 6-8 weeks at multiple points. The antigen inoculation dose for each mouse is 50 μg. After three weeks, mix the antigen and Freund's incomplete adjuvant at a ratio of 1:1 into 500 μL, emulsify and subcutaneously inject at multiple points. The antigen inoculation dose for each mouse is 50 μg. After an interval of three weeks, 50 μg should be injected intraperitoneally without adjuvant for total immunization. 4 times. The results are shown in Table 1:

Table 1 Result determination of mice immunized with synthetic antigen procalcitonin protein

3. Immune serum titer determination

The immune serum titer was determined using indirect ELISA. Dissolve 50 μg of the synthetic antigen procalcitonin protein in 10 mL of 0.05M pH9.6 phosphate buffer, coat it on a polystyrene 96-well plate, 100 μL/well, and incubate at 4°C overnight. Wash the plate three times with PBS (containing 0.05% v/v Tween-20), use 100 μL/well of 10mM PBS containing 1% BSA blocking solution, block at 37°C for 2 hours, and wash the plate with PBS (containing 0.05% v/v Tween-20). Three times, blood was collected from the mouse tail vein 10 days after the third immunization. The mouse immune serum was diluted 10 ^-2 to 10 ^-8 times with 10mM PBS containing 1% BSA, and added to a 96-well plate, 100 μL/well at 37°C for 1 hour, PBS (Containing 0.05% v/v Tween-20) After washing the plate three times, add 1:10000 diluted horseradish peroxidase-labeled goat anti-mouse IgG (Sigma, INC.), 100 μL/well, 37°C for 30 min, the same as above After washing the plate, develop color with TMB, 100 μL/well, protect from light at room temperature for 10 min, add 50 μL/well 2M H ₂ SO ₄ to terminate the reaction, measure the 450 nm absorption value, and use pre-immune mouse serum as a negative control to compare the measured value with the control value. A ratio of ≥2.1 is considered a positive judgment value to determine the immune serum titer.

4. Preparation of hybridomas

For mice with a serum titer greater than 1:10 ⁵ , 3 days before fusion, mix the synthesized antigen procalcitonin protein with an equal volume of PBS, and then intraperitoneally inject BALB/c at a dose of 50 μg/500 μL each. Fusion mice were boosted. Aseptically remove the mouse spleen, make a spleen cell suspension and mix it with the mouse myeloma cell line SP2/0 in the logarithmic growth phase at a ratio of 1:1, centrifuge at 1000g at room temperature for 5 minutes, discard the supernatant, and centrifuge lightly with your fingers. At the bottom of the tube, loosen the precipitate. Place the centrifuge tube in a 37°C water bath. Add 50% polyethylene glycol (PEG, MW4000, Sigma) insulated in a 37°C water bath into the centrifuge tube drop by drop with a dropper, shaking while dropping. Complete the dripping in the centrifuge tube within 1 minute. After the dripping is completed, let it stand for 2 minutes. Add 1mL, 2mL, 3mL, 4mL, 5mL and 10mL of serum-free 1640 medium preheated at 37°C every 1 minute to terminate the effect of polyethylene glycol. The mixture was centrifuged at 1000g for 5 minutes at room temperature, the supernatant was discarded, and HAT culture medium (hypoxanthine (H), aminopterin (A), and thymidine (T) (HAT, Sigma)) was added to gently resuspend the cells, and the cells were separated. into a 96-well plate, 200 μL per well. After three days of culture, the cell fusion was observed, and half of the HAT culture medium was replaced for several days until colonies were formed. Seven days after fusion, the HT culture medium (hypoxanthine (H) and thymidine (T)) was replaced (HT, Sigma ))nourish.

5. Screen hybridoma cells secreting anti-procalcitonin monoclonal antibodies

Screen the cell culture supernatant by indirect ELISA, select positive cloned hybridoma cells with higher potency for subcloning (Table 2), and use the limiting dilution method for continuous cloning 2-3 times until the cell positivity rate is 100%, and finally A cell line that stably secretes anti-procalcitonin monoclonal antibody was obtained, labeled A4D7, etc. The cells with a positive rate of 100% after cloning were expanded and cultured and then frozen in liquid nitrogen.

Table 2 Monoclonal antibody screening results

6. Preparation and purification of ascites

The hybridoma cell line A4D7 was injected into the peritoneal cavity of 8-10 week old female BALB/c mice pretreated with liquid paraffin at a volume of 1×10 ⁶ /mouse. After 10-14 days of feeding and observation, the abdominal fluid was extracted when the abdomen of the mice was swollen. The monoclonal antibody was purified by affinity chromatography Protein G Sepharose Fast Flow, and the purity of the monoclonal antibody was determined by SDS-PAGE, which was more than 90%.

Example 2 Characterization of monoclonal antibody A4D7

1. Determination of antibody concentration: Ascites prepared by hybridoma cells A4D7, etc. are purified to obtain anti-procalcitonin monoclonal antibodies A4D7, etc., and measured using the Nanodrop nucleic acid protein analyzer produced by Thermofisher Company. The concentration is >1mg/ ml.

2. Antibody subtype identification: Use Thermofisher's mouse monoclonal antibody subtype identification kit to identify the subtype of the hybridoma cell line. The subtype of the antibody secreted by A4D7 is IgG1 type, and the light chain is κ chain.

3. Validation of purified antibodies: 50 μg of synthetic procalcitonin protein was dissolved in 10 mL of 0.05 M carbonate coating buffer at pH 9.6, added to a 96-well plate, 100 μL per well, and kept overnight at 4°C. Wash the plate three times with PBS (containing 0.05% v/v Tween-20), use blocking solution (150 μL/well) of 10mM PBS containing 1% BSA, block for 2 hours at 37°C, and use PBS (containing 0.05% v/v Tween-20) Wash the plate three times, add 100 μL of purified antibody to each well, incubate at 37°C for 1 hour, wash the plate three times with PBS (containing 0.05% v/v Tween-20), and add horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody as diclonal antibody. Antibody, incubate at 37℃ for 30min, wash the plate three times with PBS (containing 0.05% v/v Tween-20), add 100μL to each well, develop color with TMB, after incubating at 37℃ for 15min, add 2M H ₂ SO ₄ solution to terminate the reaction, enzyme label The instrument detects the absorbance value at 450nm.

4. Antibody binding capacity test:

Dilute procalcitonin protein with 1×CB to 0.5 μg/mL and 1 μg/mL respectively, add 100 μL/well into the wells of the enzyme plate, make duplicate wells, and place at 4°C overnight or adsorb at 37°C for 2 Hour. Spin the coated microplate dry, wash it once according to the operating program (AFP program) set by the plate washer, add blocking solution at an amount of 200 μL/well, place it in a 37°C incubator for 2 hours, and then place it at 4 °C overnight. Before use, take out the sealed microwell plate from 4°C, spin it dry, add cleaning solution (1×PBS-T) to moisten the enzyme plate; pre-dilute the monoclonal antibody A4D7 of the present invention with 1×PBS. to 30 μg/mL, and record the pre-dilution multiple m, then dilute it 10 times, that is, 3 μg/mL as the highest concentration (S1), and then dilute it with a 1:3 gradient (diluted in a 96-deep well plate), a total of 8 dilutions Gradient (S1-S8).

Add 100 μL of diluted antibody to a 96-well microplate patted clean on absorbent paper, and incubate at 37°C for 30 minutes; after incubation, spin the enzyme plate dry, pat it dry on absorbent paper, and clean the enzyme plate with a plate washer. Three times, add 100 μL of 1×PBS to each well of columns 1-4; add 200 μL of urea treatment solution to each well of columns 5 and 6, and incubate at 37°C for 30 minutes; after incubation, spin the enzyme plate dry and pat dry on absorbent paper. , wash the enzyme-labeled plate 3 times with a plate washer, add 100 μL of GAM-HRP enzyme-labeled secondary antibody that has been diluted 10,000 times with secondary antibody diluent to each well, and incubate at 37°C for 30 minutes; after incubation, spin-dry the enzyme-labeled plate and absorb water. Pat the paper dry, wash the enzyme plate three times with a plate washer, take TMB chromogenic solution, add 100 μL of chromogenic solution to each well, and incubate at 37°C for 5-10 minutes; after color development, add 50 μL of stop solution to each well. Set the microplate reader to read at 450nm/630nm.

The ELISA antigen-antibody binding experiment was tested under the conditions of 0.5μg/mL and 1μg/mL antigen coating, respectively. The data were obtained by testing the absorbance OD (Figure 2), and the corresponding polynomial curve was fitted. The corresponding binding showed an obvious gradient with the change of the antibody dilution ratio. At the same time, the affinity binding reflected by the two curves under the conditions of 0.5μg/mL and 1μg/mL antigen coating reflected the specificity of the binding and reflected that the concentrations corresponding to the antibody binding force reached 6.56E-10mol/L (under the conditions of 0.5μg/mL antigen coating, the highest OD reading was 50%, that is, when the concentration of the antigen-antibody complex accounted for half of the total concentration, the corresponding concentration value of the antibody was K _0.5 ) and 1.28E-09mol/L (under the conditions of 1μg/mL antigen coating, the highest OD reading was 50%, that is, when the concentration of the antigen-antibody complex accounted for half of the total concentration, the corresponding concentration value of the antibody was K ₁ ).

Binding force calculation: Calculate the average OD readings of the multiple wells of S1-S8 antibody concentrations before and after urea treatment under 0.5 μg/mL and 1 μg/mL antigen coating conditions. Substitute the reading value into the binding force calculation formula:

5. Monoclonal antibody pairing screening experiment:

The purified A4D7 monoclonal antibody was used to complete the coupling work. It was matched with three other procalcitonin monoclonal antibodies, the clone numbers of which were F4B4, A8E5, and C6H1. 49 clinical samples were tested using the matched products and the test results were obtained. The results were analyzed by Bland-Altman curve as follows:

Through curve analysis, it can be seen that when A4D7 is combined with F4B4 for sample testing, the test results have the highest correlation with the background value of the serum sample. Therefore, A4D7 and F4B4 are the best combination (Figure 3, A-C).

6. Antibody stability verification:

The experimental operation method is as follows:

6.1 Take 2mg of antibody A4D7, divide it equally into 4 pieces of 0.5mg/tube, and seal it with a sealing film.

6.2 Take one of them for processing and coupling to make a reagent. Save the reagents.

6.3 Randomly pick one of them and freeze it, and store the other one in a 37°C incubator. The storage time is 2 days (48 hours).

6.4 After storage, return the antibody to room temperature.

6.5 Prepare the antibodies according to the process. After the preparation is completed, use the samples to test the three experimental groups.

Compare the test results of control storage methods, frozen storage and storage at 37°C. The stability of the antibodies as well as the stability of the reagents prepared using the antibodies showed good results (Figure 4).

7. Monoclonal antibody A4D7 sequencing

Take the purified hybridoma cell A4D7 and prepare the procalcitonin monoclonal antibody A4D7. Sequencing of the light chain variable region and the heavy chain variable region revealed that the amino acid sequences of the light chain and heavy chain of antibody A4D7 are shown in SEQ ID NO: 1 and 2 respectively. The light chain variable region sequences of the above-mentioned procalcitonin-targeting antibodies are CDR-L1 (RSSDSLLHSNGITYLF), CDR-L2 (QMSALAS) and CDR-L3 (AQNLHLPWT), and the heavy chain variable region sequence is CDR-H1 (SDYSWE) , CDR-H2 (YIDFRGSTKYNPSLRS) and CDR-H3 (RGSFRY). Antibodies with corresponding variable region sequences have high affinity and good specificity; therefore, the corresponding antibody variable regions can be used for the development of recombinant antibodies, single-chain antibodies, and bispecific antibodies, and for the development of related products for diagnostic or therapeutic purposes. .

Example 3 Procalcitonin lateral flow immunochromatography reagent performance test

Immuno(fluorescence) microspheres are one of the labeling materials used in immunochromatography detection kits. By modifying the carboxyl terminus on their surface, they can covalently bind to biological macromolecules such as proteins with amino termini. Through this gentle coupling, the activity of biological macromolecules can be retained to the maximum extent. The above advantages make immune (fluorescent) microspheres become the labeling material in commercial immunochromatography test strips widely used on the market.

The lateral flow immunochromatography rapid diagnostic kit is a test strip-based result reporting method suitable for point-of-care testing. The core component is the lateral flow immunochromatography test strip, which is generally in the shape of a narrow strip, usually with a width of 4-6 mm and a length of no more than 6-7 cm. A standard lateral flow immunochromatography test paper should include four main parts, namely: the main material of the sample pad area is made of cellulose. When using, the sample pad area should be fully immersed in the substance to be tested; the binding pad area The main material is made of glass fiber, and the binding pad area generally contains (coupled) antigen or (coupled) antibody; the main material of the detection area is made of nitrocellulose membrane (NC membrane), and there are mainly detection lines and The quality control line, detection line and quality control line respectively contain corresponding antibodies; the main material of the absorbent pad area is made of cellulose.

When the lateral chromatography test paper is inserted into the solution to be measured, the solution will flow from the sample pad area into the binding pad area. When the solution contains a substance to be detected, the substance to be detected will bind to the (coupled) antigen or (coupled) antibody in the binding pad area. The (coupled) antibody or (coupled) antigen or the complex formed with the sample to be tested will pass through the detection area (NC membrane) and the water-absorbent pad area in sequence under the action of capillary force. At this time, the lateral flow test paper will have two result display models, namely the standard model and the competition model:

When the lateral flow immunochromatographic test paper adopts the standard model (detecting PCT protein antigen), the test line (including the specific antibody targeting PCT protein antigen) can only capture the complex of the antigen to be tested and the (coupled) primary antibody: if the antigen to be tested exists in the solution, the test line can capture the complex formed by the antigen to be tested and the (coupled) primary antibody, and because the (coupled) primary antibody itself will always be captured by the quality control line (including the corresponding secondary antibody), the color of both the test line and the quality control line will change at this time, and the result is judged as a positive reaction. If the antigen to be tested does not exist in the solution, only the quality control line (including the corresponding secondary antibody) can capture the colloidal gold (coupled) primary antibody and change color. At this time, the quality control line will be colored but the test line will not be colored, and the result is judged as a negative reaction.

When the lateral flow test paper adopts the competition model (detecting IgG antibodies targeting PCT protein antigen), the antibody to be tested binds to the (coupled) PCT protein antigen coated in the binding pad area, and the antibody to be tested binds to the colloid. The gold (conjugated) PCT protein antigens can each be captured by the detection line (containing anti-IgG antibodies): when the antibody to be tested is present, it can be captured by the detection line and the (conjugated) antigen will not be captured by the detection line. (Because of the competitive blocking of the antibody to be tested), the color change cannot occur, but the quality control line (including the PCT protein antigen paired antibody) can still capture (couple) the antigen. At this time, the detection line does not develop color but the quality control line does. color, the result is judged as a positive reaction; if there is no antibody to be tested in the solution, both the test line and the quality control line can capture (couple) the antigen, and at this time both the test line and the quality control line will change color, and the result is judged as Negative reaction.

After the antibodies (A4D7 and F4B4) were paired, the (semi-) quantitative relationship evaluation of the antibodies targeting the purified procalcitonin protein was carried out by the above method: the targeted reduction antibody produced by Hangzhou Huakui Jinpei Biotechnology Co., Ltd. was used. The quality control products of procalcinogen protein antibodies, (Roche) antigen products, etc. were respectively tested on the lateral flow immunochromatography quick test kit developed for the antibodies of the present invention. After using specific instruments (FIC-Q1) and antibodies ( On the premise of specific chemical labeling (antibodies targeting procalcitonin), time-resolved fluorescence immunoassay is used to relatively accurately estimate the concentration of labeled antibodies within a certain concentration range (and compared with homogenized products from other companies for estimation Accuracy of quantitation of labeled antibody concentration). Since the target antigen PCT and the paired antibodies are both purified protein products developed by the company, they can be used to evaluate the accuracy of the quantitative relationship within a certain concentration range. From the comparison in Table 3, it can be seen that the relationship between the antibody concentration estimated by comparing the curve fitting of the quality control product (QC-product-based Ab-concentration estimation, or "QC-based Ab-con") and T/C, and the directly measured The relationship between the antibody concentration (Ag-background-based Ab-concentrationestimation, or "Ag-based Ab-con") and T/C estimated by the antigen (Roche) background, as well as the fitting curve obtained from the fixed parameters recommended by the finished product box. Comparison of the relationship between antibody concentration (Product-fixed-model-based Ab-concentration estimation, or "Product-based Ab-con") and T/C, etc. The results suggest the antibody concentration estimation method based on the fixed parameters recommended by the finished product box And the method of estimating the antibody concentration through the estimation of the antibody concentration of the quality control product, compared with the directly measured antigen background, can maintain a stable linear relationship within a larger concentration range, thereby achieving relatively more accurate quantitation (without batch separation) The analysis results are shown in Figure 5, and the analysis results of batches are shown in Figure 6).

Table 3 Correlation analysis table

^* p＜.05, ^＊＊ p＜.01, ^＊＊＊ p＜.001

Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims (10)

1. Antibody A4D7 targeting procalcitonin, characterized in that the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain hypervariable region of the antibody are RSSDSLLHSNGITYLF, QMSALAS and AQNLHLPWT respectively, and the antibody M1301 The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain hypervariable regions are SDYSWE, YIDFRGSTKYNPSLRS and RGSFRY respectively. 2. The antibody according to claim 1, characterized in that the amino acid sequences of the light chain and heavy chain of the antibody are shown in SEQ ID NO: 1 and 2, respectively. 3. Application of the antibody according to claim 1 or 2 in the preparation of procalcitonin detection reagents or kits. 4. A procalcitonin detection reagent or kit prepared from the antibody of claim 1 or 2. 5. Application of the antibody of claim 1 or 2 in the preparation of procalcitonin immunochromatographic detection test strips or kits. 6. Application of the antibody of claim 1 or 2 in the preparation of procalcitonin lateral flow immunochromatography diagnostic kit. 7. Procalcitonin immunochromatography detection test strip, characterized in that it includes a sample pad, a binding pad, an NC membrane, an absorbent pad and a bottom plate. The reaction membrane is provided with a detection line and a quality control line. The sample The pad, bonding pad, NC film and absorbent pad are pasted on the base plate in sequence; Wherein, the binding pad is coated with immune microsphere-coupled antibody F4B4 targeting procalcitonin; the detection line is coated with the antibody of claim 1 or 2, and the quality control line is coated with sheep Anti-mouse polyclonal antibodies; The amino acid sequences of the light chain and heavy chain of antibody F4B4 are shown in SEQ ID NO: 3 and 4 respectively. 8. The test strip according to claim 7, characterized in that the concentration of the antibody A4D7 coated on the conjugate pad is 10-15 μg/mL; The concentration of antibody A4D7 coated on the detection line is 1.5-2mg/mL. 9. The test strip according to claim 7 or 8, characterized in that the material of the sample pad and the water-absorbent pad is cellulose, and the material of the bonding pad is glass fiber. 10. A qualitative and semi-quantitative rapid detection method for procalcitonin, characterized in that the detection method is a double-antibody sandwich method, and the amino acid sequences of the light chain and heavy chain of the first antibody used are as follows: SEQ ID NO: 3, As shown in 4, the second antibody used is the antibody described in claim 1 or 2.